Proteins represent an important and growing class of therapeutic compounds. Many vitally important therapeutic compounds for treating human and animal diseases and other conditions are currently in the market place and many more are in development. One challenge presented by proteins in general and proteins used as therapeutics in particular is obtaining an accurate understanding of their higher order structure (HOS), and ensuring that this HOS remains unchanged throughout the development and commercialization lifecycle of the drug. Examples include determining the impact of changes to manufacturing, shipping, or storage conditions on the higher order structure of proteins. Still other examples include determining the structural similarities between proposed biosimilar therapeutic compounds and the putatively bio-equivalent approved protein therapeutic.
However, the inherent complexity of protein structures presents challenges that must be addressed in order to accomplish high resolution analysis of higher order structures of proteins. Various methods have been developed in an attempt to overcome these challenges. These challenges increase exponentially when subtle changes in the protein's structure can affect its biological properties and when cost is factored into the process.
Aspects of the invention disclosed herein, seek to address these challenges.
A first set of embodiments includes methods for detecting changes in the higher order structure of proteins, comprising the steps of treating a reference protein with a first compound, under a defined set of conditions, wherein the treating step produces a covalently labeled reference protein; contacting a target protein with the first compound, under the defined set of conditions, wherein the contacting step produces a covalently labeled target protein, wherein the reference protein and the target protein have identical primary structures; and analyzing the covalently labeled target protein and the covalently labeled reference protein by mass spectrometry.
A second set of embodiments includes the methods according to the first set of embodiments, further including the steps of comparing the results of the analysis of the covalently labeled reference protein and the covalently labeled target protein; and concluding that there is a difference in the higher order structure of the reference protein and the target protein if a difference is detected in the comparing step.
A third set of embodiments includes the methods according to the first through the second set of embodiments, wherein the reference protein and the target protein includes at least one amino acid selected from the groups consisting of cysteine, histidine, lysine, tyrosine, serine, threonine, aspartic acid, and glutamic acid.
A fourth set of embodiments includes the methods according to the first through the third set of embodiments, wherein the target protein is selected from the group of proteins consisting of antibodies, enzymes, ligands, or regulatory factors.
A fifth set of embodiments includes the methods according to the first through the fourth set of embodiments wherein the reference protein has not been exposed to the same processing or the same manufacturing steps as the target protein.
A sixth set of embodiments includes the methods according to the first through the fourth set of embodiments, wherein the target protein has been stored in a suspension buffer designed to stabilize the reference protein, or in a lyophilized form for a period of time longer than the time that the reference protein has been stored in the suspension buffer or in a lyophilized form.
A seventh set of embodiments includes the methods according to the sixth set of embodiments, wherein the suspension buffer include at least one claims of reagent selected from the group of reagents comprising: phosphate, amino acids, inorganic salts, surfactants, metal chelators, polymers, inert proteins, and preservatives.
An eighth set of embodiments includes the methods according to the sixth through the seventh set of embodiments, wherein the suspension buffer has a pH in at least one pH range selected from the group consisting of, between about 2.0 to about 10.0; between about 2 to about 9.0; between 3 to about 10.0; between 3 to about 8.0; between about 3.5 to about 7.5; between about 4.5 to about 6.5; and between about 5.5 to about 7.3.
A ninth set of embodiments includes the methods according to the sixth through the eighth sets of embodiments, wherein the suspension buffer includes at least one of the following amino acids selected from the group consisting of histidine, arginine, glycine, methionine, proline, lysine, glutamic acid, alanine, and arginine mixtures.
A tenth set of embodiments includes the methods according to the sixth through the ninth sets of embodiments, wherein the suspension buffer includes at least one of the following inorganic salts selected from the group consisting of sodium chloride, calcium chloride, and magnesium chloride.
An eleventh set of embodiments includes the methods according to the sixth through the tenth sets of embodiments, wherein the suspension buffer includes at least one of the surfactants selected from the group consisting of polysorbates, SDS, Brij 35, and Triton X-10.
A twelfth set of embodiments includes the methods according to the sixth through the eleventh sets of embodiments, wherein the suspension buffer includes EDTA as a metal chelator.
A thirteenth set of embodiments includes the methods according to the sixth through the twelfth sets of embodiments, wherein the suspension buffer includes at least one of the following polymers selected from the group consisting of polyethylene glycols (PEGs) and polysaccharides.
A fourteenth set of embodiments includes the methods according to the sixth through the thirteenth sets of embodiments, wherein the suspension buffer includes at least one of the following inert proteins selected from the group consisting of dextran, hydroxyl ethyl starch (HETA), PEG-4000, and gelatin.
A fifteenth set of embodiments includes the methods according to the sixth through the fourteenth sets of embodiments, wherein the suspension buffer includes at least one of the following preservatives selected from the group consisting of benzyl alcohol, m-cresol, and phenol.
A sixteenth set of embodiments includes the methods according to the first through the fifteenth sets of embodiments, wherein the compound used to label the reference protein and the target protein is diethylpyrocarbonate.
A seventeenth set of embodiments includes the methods according to the first through the sixteenth sets of embodiments, wherein the proteins being labeled are proteins with a molecular weight of at least 5 kDa.
An eighteenth set of embodiments includes the methods according to the first through the sixteenth sets of embodiments, wherein the proteins being labeled are proteins with a molecular weight of at least 12 kDa.
A nineteenth set of embodiments includes the methods according to the first through the eighteenth set of embodiments, wherein the proteins being labeled are therapeutic proteins.
A twentieth set of embodiments includes the methods according to the first through the eighteenth set of embodiments, wherein the proteins being labeled are monoclonal antibodies.
A twenty-first set of embodiments includes the methods according to the first through the twentieth set of embodiments, further including the step of determining the fraction of the amino acids in the target protein that are labeled as a function of the concentration of the protein and/or the concentration of the compound in the contacting step. In some of these embodiments the compound is DEPC.
A twenty-second set of embodiments includes the methods according to the first through the twenty-first set of embodiments, wherein in the fraction of the amino acids in the target protein modified by the compound is determined as a function of the time that the target protein and the compound are in contact with one another. In some of these embodiments the compound is DEPC.
A twenty-third set of embodiments includes the methods according to the first through the twenty-second set of embodiments, wherein one or more of the proteins in the assay has undergone partial degradation or denaturing.
A twenty-fourth set of embodiments includes the methods according to the first through the twenty-third set of embodiments, wherein the onset and growth of protein aggregates is monitored by % labeling at one or more amino acids where % labeling correlates with aggregation.
A twenty-fifth set of embodiments includes a means for comparing the HOS of proteins, comprising the steps of labeling a reference protein with a covalent label, to form a labeled reference protein; tagging a target protein with the covalent label, to form a labeled target protein, wherein both the reference protein and the target protein are treated with at least one reagent that covalently labels the proteins; analyzing both the labeled reference protein and the labeled target protein by use of the same mass spectrometry; and comparing the mass spectra of the labeled reference protein and labeled target protein to one another, wherein said reference protein and said target protein are substantially similar to one another.
A twenty-sixth set of embodiment includes the means according to the twenty-fifth set of embodiments, wherein the reagent that covalently labels the reference protein and the target protein is diethypyrocarbonate.
In some embodiment of the invention a sample of a protein in its unaltered state is digested and analyzed to determine the peptide map. Digestion consists of combining and incubating the protein with a preotolytic enzyme, such as trypsin or chymotrypsin. The enzyme is quenched, and, after workup, the peptides analyzed via mass spectrometry.
In some embodiments a sample of the protein in its unaltered state (ie—the reference protein) is then covalently modified. Modification may include first identifying covalent labels most suitable for labeling the protein of interest based on the amino acid makeup of the protein. If more than one covalent label is to be used, each may be combined with the protein separately, or combined with the protein at the same time. A sample of the protein is combined with the covalent label in an appropriate buffer solution. In some embodiments of these methods, samples are collected from the solution as a function of time so that the % incorporation of the label can be tracked. In other embodiments of this method, multiple sample preparations will occur, with the relative concentrations of the protein and covalent label varying in each preparation, and samples collected from each of the preparations after the same elapsed reaction time. In this case, the % incorporation of the label can be tracked as a function of covalent label concentration. This approach is often used when the covalent label can degrade in the reaction solution, such as when an anhydride label is used in an aqueous buffer solution. Each sample is digested and analyzed via mass spectrometry as described above. Comparison of the mass spectrometry results from the covalent labeling experiments vs. the initial peptide mapping experiments will allow for identification of the residues that are modified by the covalent label, and the extent of modification at each residue as a function of time and/or concentration. The results from multiple labels can be combined to give a more complete description of the overall protein HOS.
In some embodiments a sample of the target protein is then subjected to the same covalent labeling method as described for the reference protein. The mass spectrometry results for the target protein can then be compared to the reference protein, with the location of labeling, and the extent of labeling as a function of time and/or concentration, compared. Changes in the location and/or % incorporation indicate a change in the HOS structure of the target protein vs. the reference protein.
Some embodiments of the invention include methods for determining the higher order structure of proteins, comprising the steps of: contacting at least a portion of a target protein with a covalent label in order to produce a covalently labeled target protein; modifying a reference protein with the same covalent label in order to produce a labeled reference protein; analyzing the covalently labeled target protein and the labeled reference protein by use of the same mass spectrometry technique; and comparing the results of the analysis of the covalently labeled target protein to a reference protein, in order to determine if there is a detectable difference between the labeled target protein and the labeled reference protein.
In some embodiments of the invention the target proteins includes at least one amino acid selected from the groups consisting of cysteine, histidine, lysine, tyrosine, serine, threonine, aspartic acid, and glutamic acid. In some embodiments a single covalent label is used to create the covalently labeled target protein. In other embodiments two or more different covalent labels are used to create the covalently labeled target protein, and wherein the results of the individual covalent bond analyses are combined to increase the fraction of amino acids in the target proteins that are measured in a given assay.
In some embodiments the methods further include the step of determining the fraction of the amino acids in the target protein that are labeled as a function of the concentration of the protein and/or the concentration of covalent label modifier in the contacting step. In some embodiments the fraction of the amino acids in the target protein modified by the covalent labels is determined as a function of the time that the target protein and at least one covalent label are in contact with one another. In some embodiments the inventive methods are carried out using proteins, especially target proteins, that may have undergone partial degradation or denaturing.
Still other embodiments of the invention include means for comparing the HOS of proteins, comprising the steps of: labeling a reference protein with a covalent label, to form a labeled reference protein; tagging a target protein with the covalent label, to form a labeled target protein; analyzing both the labeled reference protein and the labeled target protein by use of the same mass spectrometry; and comparing the mass spectra of the labeled reference protein and labeled target protein to one another, wherein said reference protein and said target protein are substantially similar to one another.